Two main host fields (alfalfa, Medicago sativa L. and mixed vegetables), located at the Agricultural Operations of the University of California, Riverside, were selected for the sampling surveys (Fig. 1). The alfalfa field was selected because it was a host for several stink bug species including Nezara viridula (Linnaeus), Chlorochroa sp., and Thyanta sp. during the spring and summer of 2017, making it a potential source of parasitoids that might parasitize B. hilaris eggs. The mixed vegetable field included bell pepper (Capsicum annuum L.), broccoli, cantaloupe (Cucumis melo L.), corn (Zea mays L.), okra (Abelmoschus esculentus L.), tomato (Solanum lycopersicum L.), and zucchini squash (Cucurbita pepo L.). Mid-way through the study (January 2018), a squash field (Cucurbita mochata L., cultivars black futsu and shishigatani) was selected when B. hilaris adults were found on shortpod mustard weeds within the field. In addition to these fields, sentinel egg cards were placed at other locations on the Agricultural Operations property where mustard weeds were found (Fig. 1).

Figure 1. 

Locations at the Agricultural Operations of the University of California, Riverside that were selected to survey for resident egg parasitoids of Bagrada hilaris. Yellow squares show the host fields that included squash: Latitude 33°57'58.08"N, Longitude 117°20'35.37"W; alfalfa: Latitude 33°57'54.20"N, Longitude 117°20'27.01"W; and mixed vegetables: Latitude 33°57'56.32"N, Longitude 117°20'25.48"W. Yellow dots show other locations where mustard weeds were found and sentinel cards were placed. The green dot represents the location where Trissolcus hyalinipennis was recovered and the red dots represent the locations where Trissolcus basalis was recovered.

Bagrada hilaris eggs (≤ 24 hours old) were collected and glued (Gorilla Super Glue Gel, The Gorilla Glue Co., Ohio, USA) on a 3 × 5 grid of squares on a weatherproof card so that each card contained 15 eggs (Fig. 2). For each location, two sentinel cards (a total of 30 eggs) were attached to the wire of a landscape flag (Fig. 3). The upper card was taped to the flag wire, positioning it about 30 cm from the ground. The lower card was designed to be folded into a triangle through which the flag wire was used to secure it to the ground (Fig. 3). The number of cards used for each survey date varied from 16–20 depending on the availability of B. hilaris eggs. Sentinel egg cards were deployed in the survey sites starting in October 2017. Cards were left in the field for a maximum of 5 days, since B. hilaris eggs hatch after 5 days at similar temperatures (Reed et al. 2017). A total of 113 cards (1695 eggs) have been deployed to date. Surveys were conducted on October 21–25, 2017, November 3–8, 2017, January 5–7, 2018, January 12–16, 2018, January 19–23, 2018, and January 26–30 (Tab. 1). In the first 2 survey times, most of the eggs on the lower cards appeared to be eaten by predators (Fig. 4, Tab. 1) and the bug hatch rate was lower for the eggs glued on the lower cards than the upper cards (Tab. 1). Therefore, the surveys for January and beyond consisted only of upper cards.

Figure 2. 

The sentinel card grids with fresh Bagrada hilaris eggs.

Figure 3. 

From left to right: The triangular and flat sentinel cards used in the study; the cards attached to a landscape flag wire in an alfalfa field; and in a vegetable field with broccoli and wild mustard.

Figure 4. 

A Bagrada hilaris parasitized egg with the hole for wasp emergence B Bagrada hilaris egg hatching C B. hilaris egg showing signs that is had been eaten by a predator. Scale bars are 0.25 mm. Photo by Roger Burks.

Once collected from the field, each grid was cut from the cards, placed in small Petri dishes (6 cm in diameter), and sealed with parafilm. The lid of the Petri dish had a 1-cm hole covered with a fine mesh screen for ventilation. The Petri dishes were maintained in the same insectary room that was used for the B. hilaris colony (30±1 °C, 40–50% humidity and 14:10 (L:D) photoperiod) and were checked daily for bug hatch or wasp emergence. Emerged wasps were transferred to vials containing 95% ethanol for identification.

The Biodiversity Science: City & Nature (BioSCAN) is an urban biodiversity project of the Natural History Museum of Los Angeles County (LACM), which is attempting to assess and understand the effects of urbanization on insects. Sampling has taken place across a large part of the urban areas of the Los Angeles Basin, the San Fernando Valley, and the San Gabriel Valley, a continuously urban area housing approximately 10 million people. In order to sample continuously in a variety of urban habitats, the project seeks volunteers (‘citizen scientists’) who agree to host a Malaise trap (lightweight Townes design) and weather station for an entire year. Specimens are captured and preserved in 95% ethanol, and sorted by staff and volunteers at the LACM.

Now entering its fourth year, the BioSCAN project has encountered an extraordinary diversity of unexpected species, including rare drosophilid flies (Grimaldi et al. 2015), at least one new mycetophilid fly (Kerr 2014), and 43 species of phorid flies new to science (Hartop et al. 2015, 2016). The project is ongoing, and analyses of the data on flies (Diptera) are progressing (Brown and Hartop 2016, Hartop et al. 2018). Yet, other insect groups have been given only cursory attention.

The recovered specimens of T. hyalinipennis were identified using recent treatments of Trissolcus in the Nearctic and Palearctic regions (Talamas et al. 2015a, Talamas et al. 2017). The DNA voucher specimen of T. hyalinipennis (DPI_FSCA 00010101) is deposited in the Florida State Collection of Arthropods (FSCA), Gainesville, FL. Specimens from the BioSCAN project are deposited in FSCA and the Los Angeles County Museum of Natural History. Vouchers of the specimens used to generate the DNA barcode database are deposited in FSCA, the National Museum of Natural History (USNM), Washington, DC, and the European Biological Control Laboratory (EBCL), Montpelier, France. Images of the specimens recovered in California, additional specimens of T. hyalinipennis, and all Nearctic and Palearctic species of Trissolcus, are available from the image database Specimage (specimage.osu.edu). Collection data associated with these specimens are deposited in the Hymenoptera Online Database (hol.osu.edu).

Genomic DNA was nondestructively isolated from the whole specimen using the Qiagen DNeasy kit (Hilden, Germany) as described in Giantsis et al. (2016). The barcode region of the mitochondrial Cytochrome Oxidase Subunit I (COI) was amplified using the universal barcoding primers LCO1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’) and HCO2198 (5’-TAAACTTCAGGGTGACCA-3’) (Folmer et al. 1994). The PCR was performed in a 30 µl reaction volume: 2 µl of DNA, 3 µl of 10X Qiagen PCR buffer containing 15 mM MgCl₂, 0.9 µl of each primer (0.3 µM each), 0.6 µl of dNTPs (25 mM each), and 0.2 µl of 5 U/µl Taq DNA Polymerase (Qiagen, Hilden, Germany). The PCR conditions were as follows: 94°C for 3 min, followed by 40 cycles of 94 °C for 30 s, 52 °C for 1 min, 72 °C for 1 min with a final extension at 72 °C for 10 min. All PCR products were electrophoresed through agarose gel (1%), then sent to Genoscreen (Lille, France) for sequencing in both directions. Sequences were assembled and edited using Bioedit version 7 (Hall 1999). The consensus sequence was checked to detect frame-shift mutations and premature stop codons, indicating the presence of pseudogenes using the online ORFfinder available at: http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi. The sequences generated from this study and those from our custom barcode database are deposited in Genbank (Tab. 2). All residual DNAs are archived at the European Biological Control Laboratory (EBCL).

All sequences were aligned using ClustalW with default settings as implemented in Bioedit. Searches for sequence similarity against our custom database were performed using the online BLASTn available at: https://blast.ncbi.nlm.nih.gov. The pairwise nucleotide sequence distances among and within taxa were estimated using the Kimura 2-parameter model (K2P) of substitution using Mega 6 (Tamura et al. 2013). The best fitting evolution model was selected using Akaike information criterion (AIC) as implemented in jModelTest 2 (Darriba et al. 2012). Phylogenies then were inferred under the optimal evolutionary model using Maximum Likelihood (ML) with Mega 6 and Bayesian inferences (BI) using MrBayes 3.2 (Ronquist et al. 2012). The nodes of confidence values (Bootstrap percentages, BP, and Posterior probabilities, PP) were mapped on the ML topology.

Five females of T. hyalinipennis were recovered from a backyard in Burbank California (34.165°N, -118.323°W). Samples were examined for five of the 18 sites for 2017, and additional recoveries are expected as the process of sorting and identifying material from this survey continues.

The five-merous clava found in Nearctic Trissolcus was used by Talamas et al. (2015a) to help separate Trissolcus from Telenomus, in which the number of clavomeres is usually four, but can be five or six. The arrival of T. hyalinipennis into the Nearctic region significantly decreases the utility of this character because females of T. hyalinipennis have only four clavomeres. Talamas et al. (2017) provided a new character with potential to separate Trissolcus from Telenomus: the presence of subacropleural and prespecular sulci, which are clearly defined in T. hyalinipennis (Figs 6, 9). This character, however, may be difficult for non-taxonomists to interpret and it has yet to be thoroughly examined in Telenomus. In the context of species that are known to attack the eggs of stink bugs in North America, these genera can be separated much more easily by the shape of the second metasomal tergite (T2) in dorsal view, which is wider than long in Trissolcus (Figs 5, 10, 13–14) and longer than wide in Telenomus (Fig. 7).

Figures 5–6. 

Trissolcus hyalinipennis, female (DPI_FSCA 00010101) 5 habitus, posterodorsal view 6 habitus, lateral view. sasu: subacropleural sulcus; T2: second metasomal tergite.

Figure 7. 

Telenomus podisi, female (DPI_FSCA 00010237), habitus, dorsal view. T2: second metasomal tergite.

The four-merous clava in females of T. hyalinipennis (Fig. 8) enables them to be separated from the vast majority of other Trissolcus, which have a five-merous clava (Figures 15, 17). Only one other species of Trissolcus, T. oobius (Kozlov) from the Palearctic region, is known to also have a four-merous clava. Males do not display this feature, requiring reliance on other characters for identification. The specimens recovered in this study key to T. erugatus in the dichotomous key of Talamas et al. (2015a), which differs from T. hyalinipennis by the presence of episternal foveae (Fig. 15). The absence of episternal foveae in T. hyalinipennis is a useful character because these foveae are present in nearly all other species of Nearctic Trissolcus. During the course of this study, specimens of T. basalis were reared from sentinel eggs of B. hilaris in the same locality where T. hyalinipennis was discovered. These specimens are unusually small because of the small size of bagrada bug eggs. The episternal foveae tend to be small and shallow in typical specimens of T. basalis (Fig. 16), and the diminutive size of the specimens that emerged from bagrada bug eggs contributes to an even weaker expression of these foveae (Fig. 17). These small specimens of T. basalis also exhibit striation on T2 that is extremely faint to absent (Fig. 14), as is found in T. hyalinipennis (Figs 5, 10). Males, which lack the diagnostic antennal clava, are thus very similar in appearance to males of T. hyalinipennis. They can be separated efficiently based on the form of the netrion sulcus, which is dorsally complete in T. hyalinipennis (Figs 6, 8–9) and incomplete in T. basalis (Figs 16–17).

The following characters found in T. hyalinipennis will separate both males and females from other Trissolcus in the Nearctic region: vertex without hyperoccipital carina (Fig. 10, compare to Fig. 11); frons without coarse sculpture (Fig. 8); female antennae with four clavomeres (Fig. 8, compare to Fig. 15, 17); netrion sulcus complete, extending dorsally to posterior margin of pronotum (Figs 6, 8–9, 15, compare to Figs 16–17); postacetabular sulcus comprised of discrete cells or punctures (Figs 6, 8–9, compare to Figs 16–17); anteroventral mesopleuron without episternal foveae (Figs 6, 8–9, compare to Figs 15–16); posteroventral metapleuron without setae (Fig. 6, compare to Fig. 12); mesoscutellum without coarse sculpture (Figs 5, 10); second metasomal tergite (T2) smooth and without prominent striae (Figs 5, 10).

We here present a link to an online multichoice key to Nearctic Trissolcus that includes T. hyalinipennis and newly documented variation in T. basalis: http://idtools.org:8080/key_server/player.jsp?keyId=62.

Figures 8–9. 

Trissolcus hyalinipennis 8 female (USNMENT01109060), head and mesosoma, anterolateral view 9 female (USNMENT01109061), mesosoma, lateral view. atc: acetabular carina; ats: postacetabular sulcus; bs: basiconic sensillum; nes: netrion sulcus; mpp: mesopleural pit; sasu: subacropleural sulcus.

Figures 10–12. 

10 Trissolcus hyalinipennis, female (USNMENT01109062), habitus, dorsal view 11 Trissolcus euschisti, female (OSUC 334007), head and mesosoma, dorsolateral view 12 Trissolcus thyantae, (OSUC 76325), head and mesosoma, lateral view. hoc: hyperoccipital carina; not: notaulus; white arrow in Fig. 12 indicates posteroventral setose area of metapleuron.

Figures 13–14. 

Trissolcus basalis. 13 male (DPI_FSCA 00009879), habitus, dorsolateral view 14 female (DPI_FSCA 00009651), habitus, dorsolateral view.

Figures 15–17. 

15 Trissolcus erugatus, female (USNMENT01197263), head and mesosoma, ventrolateral view 16 Trissolcus basalis, female (DPI_FSCA 00009651), head and mesosoma, ventrolateral view 17 Trissolcus basalis, female (DPI_FSCA 00009880), head and mesosoma, ventrolateral view. ats: postacetabular sulcus; bs: basiconic sensillum; eps: episternal foveae; nes: netrion sulcus.

Over the past decade, the integration of molecular barcoding and traditional taxonomic methods has facilitated accurate identification and counting of insect species (Hebert et al. 2016). While the barcoding itself is not sufficient for robust phylogenetic tree generation in complicated taxonomic groups such as Nearctic and Palearctic Trissolcus (Bon, unpublished data), it can provide an insightful means for disentangling species complexes and for preliminary species assignment. This is precisely the situation we have in the present study, where, after our first morphological examination, the initial male specimen collected in Riverside could be assigned to T. hyalinipennis or T. erugatus. When this study was initiated, Genbank and Barcode of Life Data Systems (BOLD) public nucleotide databases lacked reference barcode sequences of T. hyalinipennis and T. erugatus, although a multi-locus DNA based sequence phylogeny of expertly-identified Trissolcus specimens associated with pentatomids, including T. hyalinipennis and T. erugatus is in progress (Talamas and Bon, unpublished). We used the unpublished barcodes obtained for these two taxa to build a custom barcode database for comparison with the DNA barcode of the recovered specimen (Tab. 3). All of these unpublished barcodes were obtained as described hereafter. We also retrieved one barcode of Telenomus podisi (Accession number KR870964) from Genbank to be used as an outgroup for the phylogeny.

A barcode sequence of 661 base pairs (bp) was obtained from the specimen, and aligned with the nine barcode sequences of T. hyalinipennis and T. erugatus of our custom database (Tab. 3). The final alignment of 10 sequences consisted of 605 characters. To the best of our knowledge, these sequences constitute the first published barcode sequences for both T. hyalinipennis and T. erugatus. The Blast search of our query sequence against our custom database showed that the best identity score (99%) was obtained with all T. hyalinipennis whereas it was only 87% with all T. erugatus sequences (Tab. 3). Pairwise distance values within and among analyzed species are shown in Tab. 3. The genetic distances between the specimen from this study and all T. hyalinipennis specimens, which averaged 0.005 (± 0.002), were much lower than the mean pairwise distance observed between the specimen from this study and all of the T. erugatus specimens (0.145±0.017). The evolutionary model selected by jModelTest was the Tamura 3-parameter T92. ML topology obtained from the barcode dataset was identical to the one produced from the Bayesian analysis, and the phylogenetic tree resulting from the ML analysis is presented in Fig. 18. We found that the distinctiveness of the two Trissolcus species always had high node supports, and that the present specimen was clearly nested within T. hyalinipennis. Taken as a whole, the molecular results confirm the morphological identification.

Figure 18. 

Rooted phylogram depicting the relationships between all specimens analyzed based on the barcode dataset alignment. Bootstrap percentages (BP) ≥ 75 and Posterior Probabilities (PP)≥ 0.90 are indicated at nodes. Each line represents a sequenced specimen with reference to its collection identifier.